Distribution of isoenzymes of the glycogenolytic cascade in different types of muscle fibre.

Distinct isoenzyme patterns of the glycogenolytic enzymes exist in different fibre types. Fast twitch glycolytic and slow twitch oxidative fibres differ in the proportion of the two isoenzymes of cyclic AMP dependent protein kinase and in the type of phosphorylase kinase that is present. Slow twitch oxidative fibres and cardiac fibres resemble one another in these two respects, but differ in that the type I phosphorylase of cardiac muscle is absent in slow twitch oxidative fibres. In all examples, the functional differences between the isoenzymes seem to be related to the regulatory rather than the catalytic behavior of the molecules. In the case of cyclic AMP dependent protein kinase and phosphorylase kinase, it is a regulatory subunit that appears to be affected [16,23], while in the case of phosphorylase, the type I isoenzyme is known to have a five to eight-fold Ka for the allosteric activator 5' AMP [6]. However, the precise physiological significance of these differences remains to be elucidated.     

Characterization of the isolated rat flexor digitorum brevis for the study of skeletal muscle phosphorylase kinase phosphorylation

The flexor digitorum brevis skeletal muscle, a nearly homogeneous fast-twitch oxidative glycolytic fiber type, has been examined for its suitability to explore the regulation of phosphorylase kinase by multisite phosphorylation. A characterization of the adrenergic response of glycogenolytic enzymes, together with the previous data on contractile properties (Carlsen, R. C., Larson, D. B., and Walsh, D. A. (1985) Can. J. Physiol. Pharm. 63, 958-965), has demonstrated that this muscle is stably maintained for the several hours necessary for phosphorylation studies. The phosphorylase kinase in this muscle is primarily the alpha' isozyme, suggesting that the alpha versus alpha' isozyme distribution in muscle is related more to oxidative capacity than to fiber contractile characteristics. Using this muscle system, beta-adrenergic activation of phosphorylase kinase was observed to occur with concomitant phosphorylation of both the alpha' and beta subunits, with the total in the alpha' subunit being approximately 3-fold greater. Similarly, deactivation, following initial adrenergic activation, occurred concomitantly with the dephosphorylation of the two subunits. These results are compatible with the conclusions drawn from previous studies of the isolated enzyme and of the enzyme in perfused rat cardiac muscle, that both alpha' (or alpha) and beta subunit phosphorylation regulate phosphorylase kinase activity.     

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

The role of cysteinyl residues in the phosphorylase kinase activity as revealed by iodacetamide modification

In native nonactivated phosphorylase kinase [14C] iodacetamide interacts with 50 cysteinyl residues per enzyme molecule (alpha beta gamma delta)4. According to their reactivity towards iodacetamide these residues can be classified into 3 groups. The most reactive cysteinyl residues are involved in the enzyme activation caused by modification of SH-groups. The enzyme inhibition is biphasic. The fast and slow inactivation reactions follow the pseudo-first order kinetics. The rate of inactivation is increased by Ca2+. Mg-ATP effectively protects the enzyme against the inactivation and chemical modification of three SH-groups per protomer (apha beta gamma delta). The kinetics of inactivation and of the [14C] iodacetamide label incorporation demonstrate that two cysteinyl residues per enzyme protomer (alpha beta gamma delta) are essential for the enzyme activity. These residues are located near the ATP-binding site of the beta and gamma subunits of phosphorylase kinase.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

ADP-ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme

Phosphorylase kinase purified from rabbit skeletal muscle was ADP-ribosylated by hen liver nuclear ADP-ribosyltransferase. This modification, as was seen in cAMP-dependent phosphorylation, was observed only in alpha and beta subunits of the phosphorylase kinase and the latter was more rapidly modified. Analysis of the ADP-ribosylated amino acid residue sequenced in alpha and beta subunits showed that both subunits were modified at the area of the arginine residue. The Km for NAD was 0.10 mM and the pH optimum was 9.0. When the ADP-ribosylated phosphorylase kinase was phosphorylated by cAMP-dependent protein kinase, a reduction in phosphate incorporation occurred with increase in the ADP-ribosylation. ADP-ribosylation also suppressed autophosphorylation, to a lesser degree than observed with cAMP-dependent phosphorylation. The ADP-ribosylation-dependent reduction of phosphorylation resulted in a suppression of the phosphorylation-dependent activation of the phosphorylase kinase. These results together with findings of ADP-ribosyltransferase activity in the rabbit skeletal muscle [Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973-980] suggest that ADP-ribosylation participates in the regulation of the phosphorylase kinase activity through changes in the rate of phosphorylation.     

Effect of cyclic guanosine monophosphate on certain indices of muscle tissue carbohydrate metabolism during the wound process

The effect of intraperitoneal administration of cGMP (0.5 mg per animal) on carbohydrate metabolism of wound area muscle tissue was studied in experiments on rats with linear skin wounds. The content of glycogen, gluconeogenesis, activity of glycogen phosphorylase, lactate dehydrogenase and malate dehydrogenase were studied. Cyclic GMP induced a substantial activation of glycogen metabolism (elevation of gluconeogenesis, increase in the activity of glycogen phosphorylase) even the third day after the operation. The animals not given cGMP demonstrated such an activation only the fifth day following the operation. Under the effect of cGMP the activity of lactate dehydrogenase rose the third day after the operation. Thus cGMP administration to the animals with wounds leads to an earlier mobilization of energy resources thereby promoting the acceleration of wound healing.     

Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene

When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.     

Effects of previous activity on the energetics of activation in frog skeletal muscle

Effects of previous activity on the ability of frog skeletal muscle at 0 degrees C to liberate energy associated with contractile activation, i.e., activation heat (AH), have been examined. Earlier work suggests that activation heat amplitude (as measured from muscles stretched to lengths where active force development is nearly abolished) is related to the amount of Ca2+ released upon stimulation. After a twitch, greater than 2 s is required before a second stimulus (AHt) can liberate the same activation heat as a first stimulus (AH infinity), i.e., (AHt)/(AH infinity) = 1 -0.83 e-1.40t, where t is time in seconds. Caffeine introduces a time delay in the recovery of the ability to generate activation heat after a twitch. After a tetanus, the activation heat is depressed to a greater extent at any time than after a twitch. The activation heat elicited by a stimulus 1 s after a tetanus is depressed progressively with respect to tetanus duration up to 3 s. For tetani of 3, 40, and 80 s duration the postetanus activation heat is comparably depressed. The time-course of the recovery of the ability of the muscle to produce activation heat after a tetanus can be described as (AHt)/(AH infinity) = 1 -0.80 e-0.95t - 0.20 e-0.02t. Greater than 90 s is required before the posttetanus activation heat is equal to the pretetanus value. The faster phase of recovery is similar to recovery after the twitch and the slower phase may be associated with the return of calcium to the terminal cisternae from uptake sites in the longitudinal sarcoplasmic reticulum.     

Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.     

Calcium- and calmodulin-dependent phosphorylase kinase activity in porcine uterine smooth muscle

Porcine uterine smooth muscle phosphorylase kinase has been partially purified. The enzyme was activated about 1.5-2.0-fold by exogenous calmodulin. Half maximal stimulation was observed at about 100 nM calmodulin. The activation was dependent on calcium and was maximum at pH 7.5 in the range of pH from 6 to 9. This activation was completely abolished by 100 microM trifluoperazine. The result suggested that unlike slow and cardiac muscles, phosphorylase kinase of uterine smooth muscle showed similar response to calmodulin with that of fast muscle. The physiological role of the calcium and calmodulin-dependent activation of myometrium phosphorylase kinase is briefly discussed.     

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.     

The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.     

Dephosphorylation and inactivation of phosphorylase kinase: subunit specificity of rabbit skeletal muscle protein phosphatases

The dephosphorylation of phosphorylase kinase by four rabbit skeletal muscle protein phosphatases was studied. The four enzymes used were preparations of protein phosphatases C-I, C-II, H-I, and H-II. Phosphatases C-I, C-II, and H-II were obtained as homogeneous preparations using procedures previously developed. Phosphatase H-I was purified 644-fold from rabbit skeletal muscle for the purposes of this study, and was the major phosphorylase phosphatase activity in the tissue extract. Phosphatases C-I and H-I were relatively specific for removal of the beta subunit phosphate of phosphorylase kinase, this occurring at rates approximately 100 times more rapidly than the removal of the alpha subunit phosphate. In contrast, phosphatases C-II and H-II readily dephosphorylated both the alpha and beta subunits, although the alpha subunit phosphate release occurred at rates about twice that of the beta subunit phosphate. These studies show that skeletal muscle contains two phosphatases capable of acting on phosphorylase kinase, and that these have different specificities as represented by phosphatases H-I and C-I on the one hand, and phosphatases C-II and H-II on the other hand. These studies also provided unequivocal evidence that dephosphorylation of the beta subunit of phosphorylase kinase is solely involved in the inactivation of the cAMP-dependent protein kinase-activated enzyme. When autophosphorylated phosphorylase kinase was used as the substrate, the four phosphatases displayed similar general specificities as they did toward the cAMP-dependent protein kinase-activated enzyme. With none of the phosphatases examined was there any evidence that alpha subunit phosphorylation affected the rate of beta subunit dephosphorylation.     

Metabolic adaptation in phosphorylase kinase deficiency. Changes in metabolite concentrations during tetanic stimulation of mouse leg muscles.

1. Glycogen, nucleotides and glycolytic intermediates and products were measured before and during tetanus in the hamstrings-muscle groups of normal (C3H) and phosphorylase kinase-deficient (ICR/IAn) mice. 2. Phosphorylase kinase-deficient muscles contained 3-4-fold more glycogen and sustained a larger (approx. 2-fold), more rapid (11 +/- 2 ng/s faster) and more prolonged glycogenolysis during 120s tetanus despite their lack of phosphorylase a. 3. No significant change in total adenine nucleotide contents occurred during tetanus in either strain, but there was a 60-100-fold rise in IMP concentration to approx. 2mM in both strains. The initial rate of IMP formation was 6-fold more rapid (112 nmol/s per g) in phosphorylase kinase-deficient muscle. 4. Adenylosuccinate content rose to 36 nmol/g in phosphorylase kinase-deficient muscle and to 9 nmol/g in normal muscle at 45s tetanus, but then fell. 5. In phosphorylase kinase-deficient muscle, glucose 6-phosphate, a powerful phosphorylase inhibitor, was 56% of that in normal muscle. 6. The mass-action ratio of the phosphoglucomutase-catalysed reaction [glucose 6-phosphate]/[glucose 1-phosphate] was markedly lower than Keq. (approx. 17) in relaxed muscle of both strains (approx. 5-7), but rose significantly during tetanus to the value for Keq. 7. The data for IMP satisfy the criteria put forward by Rahim, Perrett & Griffiths [(1976) FEBS Lett. 69, 203-206] for a nucleotide activator of phosphorylase b: it should be present at a higher concentration in phosphorylase kinase-deficient muscle, its concentration should rise during muscle work, and it should attain a concentration comparable with its activation constant for phosphorylase b.     

Regulation of muscle phosphorylase kinase by actin and calmodulin

The activation of muscle phosphorylase kinase b by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase by actin is not due to trace contamination of actin preparations with calmodulin: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation of phosphorylase kinase by F-actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive both at pH 8.2 and at pH 6.8. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of Vmax.     

The effect of 5, 10, and 20 repetition maximums on the recovery of voluntary and evoked contractile properties

Maximal strength training has been reported to emphasize neural adaptations. The main objective of this study was to detect differences in muscle activation between 5, 10, and 20 repetition maximum (RM) sets. Fourteen subjects performed elbow flexion with 5, 10, and 20RM. Subjects were tested for maximum isometric force (maximal voluntary contraction [MVC]), twitch amplitude (peak twitch [Pt]), time to peak twitch (TPT), half relaxation time ((1/2) RT), electromyography (EMG), and muscle activation (interpolated twitch). Subjects were tested preexercise and 30 seconds, 1, 2, and 3 minutes postexercise. There were no significant differences in MVC, muscle activation, or antagonist/agonist EMG after 5, 10, or 20RM. However, greater RM did have a greater detrimental effect on twitch properties than fewer RM. Peak twitch was significantly (p = 0.004) less (32.08%) for the 20 than for the 5RM, whereas TPT shortened (p < 0.05) by 7.3 and 11.1% with 10 and 20RM vs. 5RM, respectively. Half relaxation time at 20RM was shortened (p < 0.05) by 20.6 and 25.4% compared with that at 5 and 10RM, respectively. MVC, muscle activation, and temporal twitch properties did not recover within 3 minutes of recovery. In conclusion, whereas 5RM did not produce greater muscle inactivation, twitch contractile properties were affected to a greater degree by a higher number of RM.     

Activation of phosphorylase kinase from rabbit muscle by actin and calmodulin

The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase b by actin is not due to the presence of trace amounts of calmodulin in actin preparations: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation by actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of V. F-actin has no stimulating effect on the phosphorylated form (a) of phosphorylase kinase or on the form a previously activated by proteolysis.